Leptospirosis is a global, zoonotic disease caused by members of the genus Leptospira. Although the disease is widespread with an estimated mortality rate of 59,000 people annually, leptospirosis is considered a neglected and understudied disease. The causative agent of Leptospirosis was first identified over 100 years ago, but the slow in vitro growth rate and limited genetic tools available to manipulate the genome of this spirochete have hampered the identification of virulence factors and development of a vaccine. Leptospires can be broadly divided into two groups: free-living saprophytes and infectious pathogens. The most widely used and studied species are L. biflexa (a non-infectious saprophyte) and L. interrogans (a pathogen). However, the nonpathogenic L. biflexa is more easily cultivated and more amenable to genetic manipulation than the pathogenic L. interrogans. Therefore, we have focused on L. biflexa as a model to characterize conserved genes and to understand the genus as a whole, to develop new techniques, and as a heterologous host to express pathogen-specific genes in order to characterize their function. Targeted gene inactivation, shuttle vector transformation, and transposon mutagenesis have all been successfully used in L. biflexa. To date, there are few published reports of targeted gene inactivations in L. interrogans. Transposon mutagenesis can be applied to L. interrogans but it functions at such a low efficiency that it cannot be utilized for any broad applications, such as auxotrophic screens or signature tagged mutagenesis. Since L. biflexa has a better transformation frequency than other species, we plan to optimize new techniques in this organism. Currently, we have two ongoing projects in this research area. In FY2018, we published the results of our study elucidating the contribution of a bactofilin protein to the morphology and motility of L. biflexa (1). This project identified a family of bactofilin proteins conserved throughout the Leptospira genus, indicating that these proteins arose early in the evolution of this family. One member of this protein family, LbbD, confers the optimal pitch distance in the helical structure of L. biflexa. Mutants lacking lbbD display a unique compressed helical morphology, a reduced motility, and a decreased ability to tolerate cell wall stressors. The change in the helical spacing combined with defects in motility and cell wall integrity showcase the intimate relationship and coevolution between shape and motility in these spirochetes. This work was completed in collaboration with Cindi Schwartz of the Research Technologies Branch, NIAID. Ongoing research on the peptidoglycan sacculus of wild type and LbbD mutant strains are being conducted in collaboration with Dr. Kelsi Sandoz (Laboratory of Bacteriology, NIAID) and Dr. Vinod Nair (Research Technologies Branch, NIAID). We believe that identifying and characterizing the factors that contribute to morphology in Leptospira spp. should aid in understanding the basic cellular physiology of these organisms and identify factors that may limit their ability to infect and disseminate in mammalian hosts. CRISPR/Cas systems are bacterial adaptive immune systems that target and degrade foreign DNA and in FY2018 we continued to evaluate the influence of the endogenous L. interrogans CRISPR/Cas system on the transformation efficiency of this organism. We have analyzed the 21 complete Leptospira genomes available in the NCBI database and found that the CRISPR/Cas systems are primarily encoded in the pathogenic spp. (such as L. interrogans, which is difficult to genetically transform) and largely absent from the saprophytic ones (such as L. biflexa, which is relatively easier to genetically transform). We hypothesize that the CRISPR/Cas systems may contribute to the lower transformation frequency observed in the pathogenic spp. relative to the saprophytes. Currently, we are attempting to inactivate specific cas genes in L. interrogans in collaboration with Drs. Wunder and Ko (Yale University), and monitor protein levels under different environmental conditions to assess which cues may induce the system, and to express the entire operon in L. biflexa to test its effect on transformation frequency in a saprophyte. If we are correct in our hypothesis regarding the role of the CRISPR/Cas system in limiting genetic transformation of the pathogenic L. interrogans, then successfully inactivating this system should generate a genetically tractable strain for assessing virulence factors. The long-term objectives of these projects are to use the improved strains and techniques to understand the basic physiology of leptospires and the mechanisms of infection and pathogenicity of L. interrogans. Together, these projects should help identify conserved proteins that may be potential targets for preventative measures.